No abstract

Offering self-sampling of cervico-vaginal material for high-risk human papillomavirus (hrHPV) testing is an effective method to increase the coverage in cervical screening programs. Molecular triage directly on hrHPV-positive self-samples for colposcopy referral opens the way to full molecular cervical screening. Here, we set out to identify a DNA methylation classifier for detection of cervical precancer (CIN3) and cancer, applicable to lavage and brush self-samples. We determined genome-wide DNA methylation profiles of 72 hrHPV-positive self-samples, using the Infinium Methylation 450K Array. The selected DNA methylation markers were evaluated by multiplex quantitative methylation-specific PCR (qMSP) in both hrHPV-positive lavage ( = 245) and brush ( = 246) self-samples from screening cohorts. Subsequently, logistic regression analysis was performed to build a DNA methylation classifier for CIN3 detection applicable to self-samples of both devices. For validation, an independent set of hrHPV-positive lavage ( = 199) and brush ( = 287) self-samples was analyzed. Genome-wide DNA methylation profiling revealed 12 DNA methylation markers for CIN3 detection. Multiplex qMSP analysis of these markers in large series of lavage and brush self-samples yielded a 3-gene methylation classifier ( and ). This classifier showed a very good clinical performance for CIN3 detection in both lavage (AUC = 0.88; sensitivity = 74%; specificity = 79%) and brush (AUC = 0.90; sensitivity = 88%; specificity = 81%) self-samples in the validation set. Importantly, all self-samples from women with cervical cancer scored DNA methylation-positive. By genome-wide DNA methylation profiling on self-samples, we identified a highly effective 3-gene methylation classifier for direct triage on hrHPV-positive self-samples, which is superior to currently available methods. .

Epigenetic host cell changes involved in cervical cancer development following a persistent high-risk human papillomavirus (hrHPV) infection, provide promising markers for the management of hrHPV-positive women. In particular, markers based on DNA methylation of tumor suppressor gene promoters are valuable. These markers ideally identify hrHPV-positive women with precancer (CIN2/3) in need of treatment. Here, we set out to identify biologically relevant methylation markers by genome-wide methylation analysis of both hrHPV-transformed cell lines and cervical tissue specimens. Genome-wide discovery by next-generation sequencing (NGS) of methyl-binding domain-enriched DNA (MBD-Seq) yielded 20 candidate methylation target genes. Further verification and validation by multiplex-targeted bisulfite NGS and (quantitative) methylation-specific PCR (MSP) resulted in 3 genes (, and ) that showed a significant increase in methylation with severity of disease in both tissue specimens and cervical scrapes ( < 0.005). The area under the ROC curve for CIN3 or worse varied between 0.86 and 0.89. Within the group of CIN2/3, methylation levels of all 3 genes increased with duration of lesion existence ( < 0.0005), characterized by duration of preceding hrHPV infection, and were significantly higher in the presence of a 3q gain ( < 0.05) in the corresponding tissue biopsy. By unbiased genome-wide DNA methylation profiling and comprehensive stepwise verification and validation studies using and patient-derived samples, we identified 3 promising methylation markers (, and associated with a 3q gain for the detection of cervical (pre)cancer..

BackgroundUrine poses an attractive non-invasive means for obtaining liquid biopsies for oncological diagnostics. Especially molecular analysis on urinary DNA is a rapid growing field. However, optimal and practical storage conditions that result in preservation of urinary DNA, and in particular hypermethylated DNA (hmDNA), are yet to be determined.AimTo determine the most optimal and practical conditions for urine storage that result in adequate preservation of DNA for hmDNA analysis.MethodsDNA yield for use in methylation analysis was determined by quantitative methylation specific PCR (qMSP) targeting the ACTB and RASSF1A genes on bisulfite modified DNA. First, DNA yield (ACTB qMSP) was determined in a pilot study on urine samples of healthy volunteers using two preservatives (Ethylenediaminetetraacetic acid (EDTA) and Urine Conditioning Buffer, Zymo Research) at four different temperatures (room temperature (RT), 4°C, -20°C, -80°C) for four time periods (1, 2, 7, 28 days). Next, hmDNA levels (RASSF1A qMSP) in stored urine samples of patients suffering from bladder cancer (n = 10) or non-small cell lung cancer (NSCLC; n = 10) were measured at day 0 and 7 upon storage with and without the addition of 40mM EDTA and/or 20 μl/ml Penicillin Streptomycin (PenStrep) at RT and 4°C.ResultsIn the pilot study, DNA for methylation analysis was only maintained at RT upon addition of preserving agents. In urine stored at 4°C for a period of 7 days or more, the addition of either preserving agent yielded a slightly better preservation of DNA. When urine was stored at -20 °C or -80 °C for up to 28 days, DNA was retained irrespective of the addition of preserving agents. In bladder cancer and NSCLC samples stored at RT loss of DNA was significantly less if EDTA was added compared to no preserving agents (p<0.001). Addition of PenStrep did not affect DNA preservation (p>0.99). Upon storage at 4°C, no difference in DNA preservation was found after the addition of preserving agents (p = 0.18). The preservation of methylated DNA (RASSF1A) was strongly correlated to that of unmethylated DNA (ACTB) in most cases, except when PCR values became inaccurate.ConclusionsAddition of EDTA offers an inexpensive preserving agent for urine storage at RT up to seven days allowing for reliable hmDNA analysis. To avoid bacterial overgrowth PenStrep can be added without negatively affecting DNA preservation.

DNA methylation is significantly associated with anal carcinogenesis. A methylation marker panel, including ZNF582, can identify anal cancer and HGAIN with a cancer-like methylation pattern. Validation studies are warranted to verify their potential for the screening and management of HIV+ MSM at risk for anal cancer.